In the last decade, application of genetically encoded fluorescent probes and sensors in molecular imaging has greatly improved our understanding about how specific molecules orchestrate cellular functions and how errant cells cause diseases. Green fluorescent protein and its related fluorescent proteins (FPs) have been successfully employed in a broad range of biological disciplines, reporting the distribution, abundance, dynamics, interaction and conformational changes of essential signaling molecules in time and space by engineering FP chimeras. However, engineering FP chimeras has long been limited by the large size (27 kDa) and biophysical properties of FPs, which can interfere with the evolved function of host proteins. Most importantly, FPs have proven to be difficult to use as sensors for probing complex biochemical processes with desired signal-to-noise ratio, such as protein phosphorylation and histone modifications. Besides FPs, small molecule based fluorophores are also useful tools for molecular imaging, but not for living cell imaging due to their lack of the cellular specificity. To date, several site-specific chemical labeling systems have been developed to detect proteins in living cells by targeting small molecular based fluorophores to peptides motifs or single-chain antibody (scFv). Though direct evolution efforts have tuned the binding affinity and specificity of small molecular fluorophores to peptide motifs, existing systems lack of the cellular specificity and have limitations as biosensors to report the functions of signaling molecules. Despite current advances in molecular imaging, tools are still lacking to allow multiplex imaging of the activity dynamics of multiple functional proteins or signaling pathways.
Therefore, a need exists for an improved chemical labeling system for molecular imaging that allows for monitoring of proteins of interest in a variety of biochemical processes and pathways. Surprisingly, the present invention satisfies this need and provides related advantages as well.